The Protective Effect And Mechanism Of Human CUL4A Overexpression On Hypoxia/Ischemia-reoxygenation/Reperfusion Injury

Hypoxic/ischemic injury has been implicated in many diseases, including various types of cancer, stroke and neurodegenerative diseases. Reoxygenation/reperfusion leads to the accumulation of DNA damage and therefore exacerbates hypoxic/ischemic injury. Hypoxia/ischemia-reoxygenation/reperfusion leads to the accumulation of misfolded or damaged proteins that are generally degraded by the ubiquitin proteasome system (UPS). The process is initiated by ubiquitination through a cascade of three enzymes, the E1activating enzyme, the E2conjugating enzyme and the E3ubiquitin ligase. Ubiquitinated proteins are then subjected to degradation by the26S proteasome. The ubiquitin E3ligase determines the specificity for ubiquitination substrates. Cullin RING-based E3is one of the largest ubiquitin E3families. Cullin4A (CUL4A) is a key member of the cullin family. By participating in the degradation of relevant proteins such as p53, p27and Cdtl, CUL4A is thought to be crucial in diverse cellular processes, such as tumor suppression, cell survival, DNA damage response, proliferation and genome stability. It has been reported that the expression of CUL4A can be induced by hypoxic injury. However, the effect of the elevated CUL4A expression on hypoxia-reoxygenation (H/R) injury is currently unclear. In this study, an adenoviral expression vector baring human CUL4A (hCUL4A) gene was constructed. hCUL4A was then successfully transfected to PC12cells and the rat brain. In the following studies, we explored the functions and mechanism of CUL4A in the H/R-or ischemia/reperfusion (I/R)-induced injury.Chapter1Cul4A gene expression in PC12cells was induced by hypoxia-reoxygenation injuryObjective:To investigate the response of endogenouse Cul4A gene to hypoxia-reoxygenation (H/R).Methods:We first established H/R experimental system in PC12cells according to the previouse report. After detecting the partial pressure of oxygen (pO2) in the hypoxic media and detecting the protein expression of hypoxia-inducible factor-1alpha (HIF-1α)in hypoxic cells to confirm the efficiency of the hypoxic procedure,PC12cells were subjected to1.5h of hypoxia followed by0.5,1,2,3,4or8h of reoxygenation. Then total RNA was isolated and semi-quantitative RT-PCR was performed to detect endogenous Cul4A expression.Results:The pO2was elevated in the media without cells after H/R. HIF-la was expressed in PC12cells subjected to3h of hypoxia. The semi-quantitative RT-PCR results shown that the expression of endogenous Cul4A increased up to3-fold during the first1-4h after H/R and then declined to normal levels8h after H/R.Conclusion:The hypoxic system is effective and stable. Cul4A gene was elevated after H/R. These results suggest the involvement of the Cul4A gene in the response to H/R in PC12cells.Chapter2Construction of the human CUL4A recombinant adenovirus vector and its expression patterns in PC12cellsObjective:To examine the function of the elevated CUL4A in the response to H/R in PC12cells, adenovirus expression vector carrying green fluorescence protein (GFP) and hCUL4A was first needed to be constructed. Then the expression patterns of the vector in PC12cells were studied.Methods:Human CUL4A gene was amplified and cloned into shuttle vector (pDC315-hCUL4A-EGFP) by In-Fusion PCR cloning technology. The skeleton plasmid of adenovirus expressioin vector, pBHG lox△E1, E3Cre, was then co-transfected with pDC315-hCUL4A-EGFP into HEK293cells using AdMaxTM vector system. After confirming the expression of hCUL4A through GFP fluorescence detection and western blot, hCUL4A adenovirus expression vector Ad-CUL4A-GFP was obtained through further viral amplification and purification. The expression patterns of hCUL4A in PC-12cells were studied after transfecting Ad-hCUL4A-GFP into PC-12cells.Results:High titer hCUL4A adenovirus expression vector Ad-hCUL4A-GFP (1.6×1012pfu/mL) was obtained. Fluorescent results shown that since the transfecting titer was more than1.6×109pfu/mL, the expression of hCUL4A-GFP could be detected after8h post-transfection. Hoechst33342staining results shown that the transfected hCUL4A mainly expressed in the cytoplasm of PC12cells. GFP fluorescence detection and western blot shown that the transfection efficiency and the expression level of hCUL4A was time and tranfectional titer dependent.Conclusion:The adenovirus vector Ad-hCUL4A-GFP was successfully constructed. That the optimal transfection titer and the spatio-temporal expression pattern of hCUL4A revealed in this study paved the way for the functional study of hCUL4A in PC12cells in the future.Chapter3The protective effects and mechanism of hCUL4A overexpression on hypoxia-reoxygenation injury in PC12cellsObjective:To investigate the functions and mechanism of hCUL4A in H/R injury in PC12cells.Methods:PC12cells were transfected by Ad-hCUL4A-GFP, then were subjected to H/R. The functions and mechanism of CUL4A in H/R were studied by detecting cell viability with WST-1, analyzing differnent stages of apoptosis with Hoechst33342staining, dealing the cells with proteasome inhibitor MG132, detecting DNA injury with single cell gel electrophoresis (SCGE), and detecting the apoptosis related protein expression (caspase3, Bcl-2, p53, p27) through western blot.Results:Cell viability was measured using the WST-1assay. Compared with the vector control group (70.94%±3.39%), cell viability increased after H/R in the hCUL4A group (87.25%±1.75%, P<0.01). Hoechst33342staining results shown that, compared with the adenoviral vector control, transfection with hCUL4A significantly suppressed H/R-induced apoptosis (24.77%±1.55%versus40.73%±2.34%, P<0.01) by decreasing the presence of stage I and stage IIa apoptotic cells. However, with the addition of the proteasome inhibitor MG132(50μmol/L), the percentage of total apoptotic cells was no longer significantly different after H/R between the two groups. Results of the SCGE shown that the percentage of DNA-damaged cells, which is determined by the percentage of nuclei with tails, was60.39%lower in the hCUL4A group than in the control group (median=11.11%versus28.05%, P<0.01) after H/R; Moreover, the tail moment decreased significantly in the hCUL4A group compared to the control group. Results of western blot shown that, H/R-induced up-regulation of caspase3activity was significantly suppressed in hCUL4A transgenic PC12cells. Consistent with the decreased caspase3activity, elevated Bcl-2levels were also observed in hCUL4A transgenic cells after H/R. Moreover, the elevated expression of p53and p27induced by H/R could be inhibited by hCUL4A overexpression.Conclusion:Overexpression of hCUL4A may protect PC12cells from H/R-induced injury by modulating apoptosis-related proteins, such as up-regulating Bcl-2, suppressing the activity of caspase3, down-regulating p53and p27. CUL4A can also suppress H/R-induced DNA damage, therefore decreasing apoptosis cells and increasing cell viability.Chapter4The protective effects of hCUL4A overexpression on ischemia-reperfusion injury in the rat brainObjective:To explore the functions of hCUL4A in ischemia-reperfusion (I/R)-induced brain injury in rat.Methods:Adenovirus Ad-hCUL4A-GFP was infused into rat brains by intracerebroventricular injection. After confirming the hCUL4A gene expression and protein expression in the rat brain3days post adenoviral injection, the middle cerebral artery occlusion (MCAO) model in rat was performed on this day. TTC staining for the assessment of the brain infarct volume and brain edema was performed at24h after MCAO/reperfusion. The histopathology of cortical neurons in the ischemic core and in the ischemic penumbra was examined3days after reperfusion using H&E staining. A widely used5-point NSS system was used to evaluate neurological behaviors from2to72h after MCAO/reperfusion.Results:TTC results shown that compared with the adenoviral vector control group (median=410.60mm3), the brain infarct volume caused by transient MCAO in the hCUL4A group (median=187.42mm3, P<0.01) was significantly decreased by54.35%. Brain edema was also reduced in the hCUL4A group compared to the vector group (median=1.069vs.1.110, P<0.05). In H&E staining brain slides, compared with the vector group, a reduction of brain edema was observed in rats in hCUL4A group, and a few red neurons and ghost cells were found. Most of the neurons were undergoing swelling, instead of the necrosis observed in the vector groups. Moreover, the proliferation of astrocytes and an increase in capillary density (up to1.7-fold vs. vector group), were also found in the hCUL4A group. NSS score results shown that compared with the vector group, significant decreases of the NSS in the hCUL4A group were observed at24,48and72h after reperfusion.Conclusion:After I/R in rat, hCUL4A overexpression can dramatically decrease brain edema and brain infarct volume, might suppress neuron necrosis and promote the repair of neural tissue, therefore promoting neurological functional recovery.In summary, our results reveal the protective effects of hCUL4A in PC12cells and rat brain upon hypoxia/ischemia-reoxygenation/reperfusion injury. Diseases such as stroke are typically characterized by hypoxia/ischemia-reoxygenation/reperfusion induced injury. Our finding raises the possibility that elevated CUL4A may exert a beneficial role in stroke disease. It will be of interest to further explore more detailed mechanisms for the protective function of the elevated hCUL4A in ischemia-reperfusion injury in in vivo stroke models or stroke disease.