ABSTRACT
Objective To explore the effects of miRNA-204 targeted LC3B expression on Ang Ⅱ induced cardiomyocytes hypertrophy.Methods The primary neonatal rat cardiomyocytes served as the research objects and divided into the control group,AngⅡ group,combination-treated group 1 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by negative control lentivirus vector),combination-treated group 2 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by lentivirus carrying miRNA-204 overexpression vector) according to different treatments.About 48 h to 72 h after intervention treatment,the cardiomyocyte hypertrophy change was detected by confocal microscopy,the expression of miRNA-204 was analyzed by real time PCR,the protein expression of LC3B was measured by Western blot and targeted gene of miRNA-204 was demonstrated by dual-luciferase reporter assay system.Results Compared with the control group,the cardiomyocyte relative surface area in the Ang Ⅱ group was significantly enlarged,the protein expression of LC3B was significantly increased,the expression of miRNA-204 was upregulated,the differences were statistically significant (P＜0.05).Whereas comparing the combination-treated group 1 with combination-treated group 2,the protein expression of LC3B in the latter was down-regulated and the cell area was reduced (P＜0.05).The further luciferase activity report gene experiment results suggested that miRNA-204 was able to bind to LC3B 3'-UTR and decreased the luciferase activities (P＜0.05),but not to bind its mutated fragment for inactivating luciferase activity(P＞0.05).Conclusion miRNA-204 is able to inhibit Ang Ⅱ induced cardiomyocytes hypertrophy,its action is realized by targeting the expression of LC3B.
ABSTRACT
AIM:To investigate the effects of exosomes secreted by pancreatic cancer cells on the viability and function of βcells and the possible mechanism .METHODS:ExoQuick-TC kit was used to extract exosomes in the super-natants of mouse pancreatic cancer Pan 02 and MPC-83 cells, and the extracted exosomes were identified by transmission electron microscopy.Fluorescence-labeled exosomes were incubated with mouse insulinoma MIN 6 cells for 48 h to detect whether exosomes secreted by pancreatic cancer cells were uptaken by MIN 6 cells.MTT and glucose-stimulated insulin se-cretion ( GSIS) assays were conducted to examine cell viability and insulin secretion of MIN 6 cells after incubating with ex-osomes.The expression of miR-204 and Bcl-2 mRNA in MIN6 cells was detected by qPCR .The protein expression of Bcl-2, Bax, caspase-3 and cytochrome C (Cyt-C) in MIN6 cells was determined by Western blot .RESULTS:The results of transmission electron microscopy showed that both Pan 02 cells and MPC-83 cells secreted exosomes , and Pan02 cells secre-ted more.The co-incubation results of fluorescence-labeled exosomes and MIN6 cells confirmed that MIN6 cells were able to ingest large amounts of exosomes secreted by pancreatic cancer cells .The results of MTT and GSIS assays showed that the viability and the level of high glucose-stimulated insulin secretion of MIN 6 cells in exosome treatment group significantly decreased compared with nontreatment group (P<0.01).The results of qPCR showed that the exosomes secreted by pan-creatic cancer cells were rich in miR-204, and the mRNA expression of Bcl-2 in MIN6 cells was significantly down-regula-ted by exosome incubation ( P<0.01) .The results of Western blot showed that the protein expression of Bcl-2 in the MIN6 cells treated with exosomes was significantly down-regulated (P<0.05), and the protein levels of Bax, cleaved caspase-3 and Cyt-C in exosomes treatment group were significantly up-regulated ( P<0.01 ) .CONCLUSION: Pancreatic cancer cells secrete exosomes .The exosomes secreted by pancreatic cancer cells are ingested by βcells, and reduce the viability and insulin secretion of βcells.The mechanism may be related to the increase in exosomal miR-204 in the βcells.In-creasing miR-204 may inhibit the expression of Bcl-2 and promote the activation of mitochondrial apoptosis in βcells.
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Objective To investigate the effect of microRNA-204(miR-204)on autophagy in U251. Methods Inhibition of miR-204 in U251 cell lines was done by transfection of miR-204 inhibitor(AMO-204) .Cell viability was detected by MTT assay.The autophagy of U251 was tested by immunofluorescence tech-nique.The protein level of Beclin 1,LC3 and Bcl-2 was detected by Western blot.Results Cell viability was markedly increased after inhibition of miR-204 in U251 cells.The number of autophagosome was decreased.The levels of Beclin 1 and LC3 were decreased,the protein level of Bcl-2 was significant increased by transfection of AMO-204 in U251 cells(P<0.05).Conclusion MiR-204 might at least in part promote glioma via inhibi-ton autophagy,indicating that miR-204 might be a potential target for the treatment of glioma.