Résumé
BACKGROUND: Studies have confirmed that microRNA (miR)-520a-3p has a regulatory role in lung cancer stem cells, but the specific function and mechanism of action are still unknown.OBJECTIVE: To investigate the influence of miR-520a-3p on the apoptosis of lung cancer stem cells, and to explore the underlying mechanism. METHODS: The magnetic activated cell sorting method was utilized to separate the CD133+lung cancer stem cells from the lung cancer A549 cell line. The expression of miR-520a-3p in the lung cancer stem cells was determined by the real-time PCR assay. Liposome transfection assay was used to up-regulate the miR-520a-3p expression level in the lung cancer stem cells, and flow cytometry assay was applied to detect the influence of miR-520a-3p expression on the apoptosis of the lung cancer stem cells. Moreover, the modulation of MAP3K2 gene by the miR-520a-3p was analyzed by the dual-luciferase reporter gene assay, and the influence of miR-520a-3p expression on the protein expression of MAP3K2, Bcl-2 and Caspase-3 was analyzed by western blot assay. RESULTS AND CONCLUSION: The real-time PCR showed that the expression level of miR-520a-3p in CD133+lung cancer stem cells was significantly lower than that in the CD133- lung cancer cells (P < 0.05). Overexpression of miR-520a-3p significantly increased the apoptotic rate of CD133+lung cancer stem cells (P < 0.05). The dual-luciferase reporter gene assay results suggested that inhibition of miRNA-520a-3p could increase the luciferase activity of the reporter plasmids containing the 3'-untranslated region (3'-UTR) of MAP3K2 gene (P < 0.05), and overexpression of miR-520a-3p could decrease the luciferase activity of the reporter plasmids containing the 3'-UTR of MAP3K2 gene (P <0.05). Moreover, miR-520a-3p overexpression also decreased the protein levels of MAP3K2 and Bcl-2 in the CD133+lung cancer stem cells, P<0.05),and increased the Caspase-3 protein level(P<0.05).To conclude,in the lung cancer stem cells,miR-520a-3p was in a low-expressed status. miR-520a-3p could inhibit the expression of MAP3K2 gene, thereby inducing the cell apoptosis.
Résumé
OBJECTIVE@#To determine the effect of tanshinone IIA on the growth and apoptosis in human hepatoma cell line HepG2.@*METHODS@#The human hepatoma cell line HepG2 was treated with tanshinone IIA at various concentrations for 72 h. The inhibition of proliferation was measured by MTT assay and apoptosis-related alterations in morphology measured by cytochemical staining (HT33258). DNA fragmentation was evaluated by agarose gel electrophoresis. Apoptotic rate and cell arrest were quantified by flow cytometry (FCM).@*RESULTS@#Tanshinone IIA inhibited the growth of HepG2 in a time- and dose- dependent manner. The semi-inhibitory concentration (IC50) value after the treatment with tanshinone IIA on HepG2 for 24, 48 and 72 h were 14.7, 7.4, and 3.9 microg/ mL, respectively. After the treatment with 0.5 - 10 microg/mL tanshinone IIA for 72 h, the formation of apoptotic bodies was observed. DNA ladder was shown in agarose gel electrophoresis, in addition to the cells treated by 1.0 microg/mL tanshinone IIA . The apoptotic rates at 0.5, 1.0, 2.0, 5.0, and 10.0 microg/mL for 72 h were 20.32%+/-2.16%, 28.0%+/-2.35%, 33.87%+/-3.43%, 46.73%+/-4.08% and 57.85%+/-3.74%, respectively, which were all significantly higher than those of the control group (P<0.05).@*CONCLUSION@#Tanshinone IIA can inhibit the proliferation of human hepatoma cell line HepG2 in a time- and dose- dependent manner, and the mechanism of growth inhibition of human hepatoma cells may be related to the induction of apoptosis.
Sujets)
Humains , Abiétanes , Antinéoplasiques d'origine végétale , Pharmacologie , Apoptose , Carcinome hépatocellulaire , Génétique , Anatomopathologie , Lignée cellulaire tumorale , Prolifération cellulaire , Fragmentation de l'ADN , Relation dose-effet des médicaments , Médicaments issus de plantes chinoises , Pharmacologie , Cytométrie en flux , Tumeurs du foie , Génétique , Anatomopathologie , Microscopie de fluorescence , Phénanthrènes , Pharmacologie , Facteurs tempsRésumé
<p><b>AIM</b>To investigate the effect of chronic lead exposure on rat hippocampal CA1 LTP and alpha-Ca2+ /calmodulin-dependent protein kinase II (alpha-CaM K II) activity in vivo.</p><p><b>METHODS</b>A stimulus bipolar electrode was placed on the Schaffer/Commissural fibers, with extra cellular microelectrode technique to record the population spike (PS) in the CA1 pyramidal, and we observed the changes of PS amplitude before and after the high frequency stimulation (HFS) of lower, mid and higher level lead exposure groups and the control group, respectively. The hippocampal CA1 alpha-CaM K II activity was determined by Western blots by using phosphorylation antibody.</p><p><b>RESULTS</b>The average changes of PS after HFS of the control group, the lower, mid and higher level lead exposure groups were 162.5%, 105.2%, 86.8%, 83.0%, respectively (P < 0.01 vs. control). Defined control a-CaM K II activity as 100, that the lower, mid and higher level lead exposure groups were 62.0 +/- 3.7, 50.8 +/- 4.0, 43.3 +/- 4.1 (P < 0.01).</p><p><b>CONCLUSION</b>Chronic lead exposure can inhibit CA1 LTP in vivo, and the decrease of alpha-CaMK II activity may play an important role in this mechanism.</p>