HIF1A基因敲除A549细胞

Tumor cells must rewire cellular metabolism to satisfy the demands of unbridled growth and proliferation. How these metabolic processes are integrated to fuel cancer cell growth remains largely unknown. Deciphering the regulatory mechanisms is vital to develop targeted strategies for tumor-selective therapies. We herein performed an unbiased and functional siRNA screen against 96 deubiquitinases, which play indispensable roles in cancer and are emerging as therapeutic targets, and identified USP29 as a top candidate essential for metabolic reprogramming that support biosynthesis and survival in tumor cells. Integrated metabolic flux analysis and molecular investigation reveal that USP29 directly deubiquitinates and stabilizes MYC and HIF1α, two master regulators of metabolic reprogramming, enabling adaptive response of tumor cells in both normoxia and hypoxia. Systemic knockout of Usp29 depleted MYC and HIF1α in MYC-driven neuroblastoma and B cell lymphoma, inhibited critical metabolic targets and significantly prolonged survival of tumor-bearing mice. Strikingly, mice homozygous null for the Usp29 gene are viable, fertile, and display no gross phenotypic abnormalities. Altogether, these results demonstrate that USP29 selectively coordinates MYC and HIF1α to integrate metabolic processes critical for cancer cell growth, and therapeutic targeting of USP29, a potentially targetable enzyme, could create a unique vulnerability given deregulation of MYC and HIF1α frequently occurs in human cancers.

Paraquat (PQ) is a bipyridine herbicide and oral exposure is the main way of PQ exposure with a very high mortality. At present, it is believed that large number of oxygen free radicals are generated and cause lipid peroxidation of tissue and organ cell membranes after PQ is absorbed. PQ exposure could cause multiple organ dysfunction, among which acute lung injury is the most common and most serious. However, its specific mechanism is still unclear. In this study, the C57BL/6J mouse (alveolar epithelial cell-specific knockout HIF-1α) model of acute lung injury (40 mg/kg PQ) at several time pointes and a model of acute type II alveolar epithelial cell (A549, 800 μM PQ) injury constructed. The oxidative stress (ROS, MDA) and inflammatory response (IL-1β, IL-6, TNF-α) were significantly inhibited in the alveolar epithelial cell-specific knockout of HIF-1α mice and siRNA technology to inhibit HIF-1α in alveolar epithelial cells. Further proteomic analysis showed that the expression of Rac2 protein, which is closely related to oxidative stress, was significantly increased after PQ exposure. And the inhibition of Rac2 expression in vitro significantly alleviated PQ-induced oxidative stress and inflammatory response. The expression of Rac2 protein was regulated by HIF-1α. The above suggests that HIF-1α may promote oxidative stress and inflammatory response in alveolar epithelial cells by regulating the expression of Rac2, and then participate in the promotion of PQ exposure-induced acute lung injury.

NARFL was reported to be a component of cytosolic iron-sulfur cluster assembly pathway and a causative gene of the diffused pulmonary arteriovenous malformations (dPAVMs). NARFL knockout dramatically impaired mitochondrial integrity in mice, which might promote mitochondrial dysfunction and lead to worse survival rate of lung cancer. However, the underlying molecular mechanism of NARFL deficiency in non-small cell lung cancer (NSCLC) is unknown. Knockdown assay was performed in A549 and H1299 cells. The protein levels of HIF-1α and DNMT1 were measured, and then Complex I activity, mtDNA copy numbers and mRNA levels of mtND genes were determined. Cisplatin resistance and cell proliferation were conducted using CCK8 assay. Cell migration and invasion were detected using wound heal assay and transwell assay. Survival analysis of lung cancer patients and KM plotter database were used for evaluating the potential value of NARFL deficiency. NARFL protein was expressed in two cell lines and knockdown assay significantly reduced its levels. Knockdown NARFL increased the protein levels of HIF-1α and DNMT1, and downregulated the mRNA levels of ND genes, mitochondrial Complex I activity, mtDNA copy number, and ATP levels. The mitochondrial dysfunction caused by NARFL deficiency were ameliorated by siHIF-1α and DNMT1 inhibitor. Knockdown NARFL increased the drug resistance and cell migration, and siHIF-1α reversed this effect. Moreover, NSCLC patients with NARFL deficiency had a poor survival rate using a tissue array and KM plotter database, and it would be a target for cancer prognosis and treatment. NARFL deficiency caused dysregulation of energy metabolism in lung cancer cells via HIF-1α-DNMT1 axis, which promoted drug resistance and cell migration. It provided a potential target for treatment and prognosis of lung cancer.