Association of T2285C polymorphism in PARP1 gene coding region with its expression, activity and NSCLC risk along with prognosis.

Poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair gene, is the crucial player in the maintenance of genome integrity. T2285C polymorphism in coding region of PARP1 has been reported to be associated with susceptibility to tumours. We explored the relationship and mechanism of T2285C polymorphism of PARP1 to its expression and activity along with risk and prognosis in non-small cell lung cancer (NSCLC). mRNA expression was measured using quantitative RT-PCR assay or collected from TCGA dataset. Protein expression was examined with immunoblotting assay. Genotypes were determined by PCR-RFLP and sequencing approaches. PARP1 activity was determined with enzyme activity assay. Regulation of SIRT7 to PARP1 was determined by overexpression and small interference experiment. Association of PARP1 T2285C polymorphism with NSCLC risk was evaluated via multiple logistic regression analysis. Comparison of treatment response and progression-free survival (PFS) of NSCLC patients among different genotypes or regimens was made by chi-square test. Results indicated that mRNA and protein expression of PARP1 dramatically increased in NSCLC tissues in comparison with paired para-carcinoma tissues (P < 0.05). TC/CC mutant genotypes were associated with markedly enhanced PARP1 mRNA level compared with TT genotype (P = 0.011). No significant difference was discovered in PARP1 protein expression among TT, TC or CC genotypes (P > 0.05). Subjects with variant allele C had higher risk of NSCLC in comparison with allele T carriers [odds ratio = 1.560; P = 0.000]. NSCLC patients carrying mutational TC or CC genotypes were correlated with unfavourable response to platinum-based chemotherapy (TT vs. TC vs. CC, P = 0.010), and shorter PFS compared with TT genotype (TT vs. TC vs. CC, P = 0.009). T2285C mutation of PARP1 resulted in the enhancement of its mRNA, but the decrease of enzyme activity in tumour cell. Overexpression of SIRT7 attenuated PARP1 expression and activity. These findings suggest the variant allele C of T2285C polymorphism of PARP1 linked to an increase of NSCLC risk, and unfavourable efficacy and prognosis of NSCLC patients with platinum-based chemotherapy, which might be associated with enhancement of its mRNA expression and the diminishment of activity. Identification of PARP1 T2285C polymorphism and mRNA expression may be the promising way for the individualised treatment of NSCLC.

SIRT7 silencing by miR-152-3p confers cell apoptosis and renal functional impairment induced by renal ischaemia/reperfusion injury.

PURPOSE: Acute kidney injury (AKI) induced by renal ischaemia/reperfusion (I/R) during renal transplantation has been reported to be linked to the regulation of SIRT2, one of the members of SIRTUINS family. Current work is attempted to explore the influence and mechanism of SIRT7 in renal cell apoptosis controlled by miR-152-3p during renal I/R injury. METHODS: Three databases were used to select the miRNAs regulating the expression of SIRT7. Overexpression and inhibition of miR-152-3p and Luciferase assay were employed to certify the modulation of miR-152-3p to SIRT7 in cells. RT-qPCR assay was used to measure the mRNA levels. Western blot assay was employed to determine the expression of proteins. TUNEL assay and Flow Cytometry were conducted to analyze cell apoptosis. RESULTS: SIRT7 expression decreased in tissues of AKI patients and rats underwent renal I/R, which was associated with enhanced impairment of renal function. SIRT7 downregulation was attributed to the direct inhibition by miR-152-3p due to binding and inhibiting its seed sequence in 3'-UTR of SIRT7 mRNA. Consequently, the upregulation of miR-152-3p led to an inhibition of SIRT7 expression, an increase in expression of extrinsic apoptosis molecules containing FOXO3a, Bim, and caspase3, and apoptotic renal cells; while miR-152-3p inhibition abolished these phenotypes. CONCLUSION: SIRT7 downregulation by miR-152-3p is a leading cause of renal cell apoptosis and functional impairment induced by renal I/R. Inhibition of miR-152-3p to restore SIRT7 expression can be a promising strategy against renal I/R injury.

SP600125, a selective JNK inhibitor, protects ischemic renal injury via suppressing the extrinsic pathways of apoptosis.

Accumulating evidence suggests that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in renal ischemia/reperfusion injury. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during renal ischemia/reperfusion has not been elucidated. We report that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion via suppression of the extrinsic pathway. This corresponds to the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3 h after renal ischemia. Additionally, SP600125 attenuated the increased expression of FasL induced by ischemia/reperfusion at 3 h. The administration of SP600125 prior to ischemia was also protective. Thus, our findings imply that SP600125 can inhibit the activation of the JNK-c-Jun-FasL pathway and protect renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for renal ischemia/reperfusion injury.

Protective effect of selenite on renal ischemia/reperfusion injury through inhibiting ASK1-MKK3-p38 signal pathway.

Previous studies have reported that selenite, a known antioxidant, protects brain against ischemia/reperfusion injury, which is mediated by oxidative stress. The aim of this study was to investigate whether selenite can protect kidney against ischemic injury by reducing activation of the apoptosis signal regulating kinase 1 (ASK1)/mitogen-activated protein kinase kinase 3 (MKK3)/p38 mitogen-activated protein kinase signaling pathway. The activation and expression of ASK1, MKK3, p38, caspase 3 and cleaved PARP were analyzed by Western blot. Apoptosis of renal tubular epithelial cells was assessed by the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling method. Malondialdehyde (MDA) levels were measured by the thiobarbituric acid reaction. Blood serum creatinine and blood urea nitrogen level were measured with an Olympus automatic multi-analyzer. We found that selenite attenuated significantly ASK1, MKK3, and p38 phosphorylation at 3 h after renal ischemia. Furthermore, selenite decreased significantly renal epithelial tubular cell apoptosis. In addition, selenite reduced the MDA level. These findings suggest that the protective action of selenite on ischemia renal injury is associated closely with reducing activation of the ASK1-MKK3-p38 signal pathway.

Protective effect of a standardized Ginkgo extract (ginaton) on renal ischemia/reperfusion injury via suppressing the activation of JNK signal pathway.

A new standardized Ginkgo extract (ginaton) destined for i.v. injection was investigated in rats for its protective effect on renal ischemia/reperfusion injury. We report on the elucidation of the downstream mechanism of action of JNK on the renal ischemia/reperfusion injury, which can be explained as the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3h after renal ischemia. At the same time, ginaton attenuated the increased expression of FasL at 3h and caspase3 immunoreactivity at 6h after renal ischemia. Furthermore, ginaton significantly decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion, alleviating renal ischemia/reperfusion injury. These results cumulatively indicate that ginaton could suppress the JNK-c-Jun-FasL-caspase3 signaling cascade, protecting renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis, which implies that antioxidants may be a potential and effective agent for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by JNK signal pathway.