RECOMBINANT ERYTHROPOIETIN MITIGATES REPERFUSION INJURY IN NEONATAL RAT CARDIOMYOCYTES BY NOVEL MULTIPLE SIGNALLING PATHWAYS

Objective: Recombinant Human Erythropoietin (rhEPO) is strongly inferred to protect the cardiomyocytes from the reperfusion injury and our aim is to elucidate the cardioprotective effect and the exact mechanism behind the cardioprotection.Methods: Neonatal rat cardiomyocytes (NCM) exposed to Hypoxia/Reperfusion (H/R) with or without pretreatment using various concentrations of rhEPO. To determine the cell viability-MTT assay, Acridine orange and Ethidium Bromide (Ao/EtBr) staining was performed. To determine the reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm), Dichlorofluorescein diacetate (DCF-DA) and Rhodamine-123 was used. To determine the signaling pathways Western blot analysis of pAkt, pp38 MAPK, cytochrome-c were performed.Results: rhEPO was found to reduce the cell death by stabilizing ROS significantly, Δψm, cytochrome c release, and caspase-3. rhEPO, increases the phosphorylation of p38 MAPK, Akt and BAD compared to H/R. Further myocytes blocked with Wortmannin (WT), and SB203580 showed increased caspase-3 activity.Conclusion: Hence we conclude from this study that rhEPO regulated the factors involved in reperfusion injury through modulation of Akt and p38 MAPK pathways

RNAI MEDIATED GENE SILENCING OF EIF3A: A POSSIBLE SOLUTION TO CONTROL BREAST CANCER

Objective: The eukaryotic translational initiation factor 3A (eIF3A) is reported to be over expressed in most breast cancer cells. In the present study, our aim is to suppress the over expression of eIF3A in human breast cancer MCF-7 cell line using gene silencing technique (RNA interference (RNAi)).Methods: The artificial microRNA (amiRNA) targeting eIF3A gene was constructed by incorporating short interference RNA (siRNA) sequences against eIF3A gene into endogenous microRNA30 (miR-30) and cloned into pcDNA3.1 vector. The amiRNA containing plasmid was then transfected into MCF-7 cell line and the expression of eIF3A was examined by RT-PCR. The cytotoxicity of plasmid with amiRNA targeting eIF3A on MCF–7 cells was evaluated by MTT assay.Results: The amiRNA construct significantly inhibited eIF3A gene expression and reduce the cell viability of MCF-7 cell line.Conclusion: The usage of modified endogenous amiRNA in vector based expression system with significant gene silencing efficiency suggests that RNAi based gene silencing method can be considered as one of the effective means to control cancer.Â