MicroRNA-210 aggravates hypoxia-induced injury in cardiomyocyte H9c2 cells by targeting CXCR4.

BACKGROUND: Myocardial infarction (MI), a leading cause of mortality, is identified as the myocardial necrosis due to prolonged ischemia. Hypoxia, resulting from ischemia, induces cell apoptosis during MI. Since miR-210 is a hypoxia inducible factor, we aimed to explore the functional role of miR-210 in hypoxic H9c2 cells. METHODS: Hypoxia-induced cell injury was evaluated according to cell viability, apoptosis and expression of apoptosis-associated proteins. miR-210 expression after hypoxia was tested. Then, miR-210 was overexpressed or silenced, and its effects on viability and apoptosis of H9c2 cells under normoxia and hypoxia were measured. Utilizing bioinformatics method, possible target genes of miR-210 were screened, and the interaction between miR-210 and target gene was investigated. Moreover, the effect of co-transfections with microRNAs and small interfering RNAs on hypoxia-induced cell injury as well as the possible involved signaling pathways was also determined. RESULTS: Hypoxia induced cell injury and up-regulation of miR-210 in H9c2 cells. Hypoxia-induced cell injury was aggravated by miR-210 overexpression but was attenuated by miR-210 suppression. CXC chemokine receptor 4 (CXCR4) was a target gene of miR-210, and CXCR4 inhibition could reverse the effects of miR-210 inhibition on H9c2 cells. Furthermore, the key kinases involved in the SMAD and mTOR signaling pathways were down-regulated by hypoxia, and the down-regulations were reversed by miR-210 suppression through modulating CXCR4. CONCLUSION: miR-210 was up-regulated in hypoxic H9c2 cells. Suppression of miR-210 attenuated hypoxia-induced cell injury in H9c2 cells by targeting CXCR4, along with activations of the SMAD and mTOR signaling pathways.

[Study on Ficolin-A against infection of Plasmodium berghei in mouse model].

OBJECTIVE: To evaluate the effect of Ficolin-A, a lectin complement against Plasmodium berghei in mice model. METHODS: The Mr 19,000 fragment of merozoite surface protein-1 of P. berghei (MSP1(19)) was cloned and then subcloned into the vector pGEX-KG. The recombinants of pGEX-KG-Ficolin-A and pGEX-KG-MSP1(19) were transformed into Escherichia coli BL21, and followed by expression of the protein induced by 1 mmol/L IPTG. The fusion protein was purified by affinity chromatography using Glutathione Sepharose 4B, and then identified by SDS-PAGE and Western-blotting. Five mouse model groups were treated with PBS, GST, Ficolin-A, MSP1(19), or Ficolin-A+MSP1(19), respectively. Each group had eight mice. Mice in Ficolin-A or MSP1(19) groups were injected with 20 microg Ficolin-A or MSP1(19) protein each time, respectively. Mice in Ficolin-A+MSP1(19) group were injected with 20 microg Ficolin-A and 20 microg MSP1(19) each time. Mice in control groups were injected with 200 microl PBS or 20 microg GST, respectively. All the mice received four immunizations at 2-week intervals. Two weeks after the last immunization, all the mice were inoculated with 300 microl Plasmodium berghei-infected red blood cells. On day 2, 4, 6, 8, and 10 post-infection, blood samples were collected from three mice of each group, and the Giemsa stained-blood films were microscopically examined. Density of malaria parasites was calculated. The survival rate was evaluated on day 20 post-infection. RESULTS: The recombinant vectors of pGEX-KG-Ficolin-A and pGEX-KG-MSP1(19) were constructed. Purified fusion proteins, Ficolin-A-GST and MSP1(19)-GST, were obtained. Western blotting analysis indicated that the relative molecular mass of fusion proteins Ficolin-A-GST and MSP1(19)-GST was about Mr 69,000 and Mr 41,000. Animal experiments showed that on day 10 after infection, the parasite density in Ficolin-A+MSP1(19) group [(22.2 +/- 1.7)%] was slightly lower than that of the groups MSP1(19) [(33.4 +/- 2.7)%], Ficolin-A [(36.2 3.1)%], GST [(43.8 +/- 4.8)%] and PBS [(45.3 +/- 3.6)%], but the difference was not statistically significant (P > 0.05). No mouse survived in PBS group on day 20 after infection. There was no significant difference in number of survival mice between Ficolin-A group (3 mice) and GST group (2 mice). Six mice survived in Ficolin-A+MSP1(19) group, which was significantly more than that of GST group (P < 0.05). CONCLUSION: Ficolin-A cannot significantly suppress parasite density. However, Ficolin-A+MSP1(19) can increase the survival rate of Plasmodium berghei-infected mice.