ABSTRACT
To investigate the role of neuropilin1 (Nrp1) in glucose metabolism and proliferation of hepatocellular carcinoma (HCC) cells and to analyze its mechanism of action. The CRISPR gene knockout technique was used to knock out the Nrp1 gene in two HCC cell lines. The effect of Nrp1 on the proliferation of HCC cells was assessed in the CCK8 assay and plate cloning assay. The expression levels of glucose consumption, lactate production, and essential proteins of the glycolytic pathway were detected to explore the effect of Nrp1 on glucose metabolism in HCC cells. Using CoCl2 to revert the expression of hypoxia inducible factor-1α (HIF-1α), the role of HIF-1α in the pro-HCC cell metabolism of Nrp1 were demonstrated. The protein synthesis inhibitor CHX and proteasome inhibitor MG-132 was used to analyze the molecular mechanism of action of Nrp1 on HIF-1α. The Kaplan-Meier method was used to calculate survival rates and plot survival curves. Based on the CCK8 assay and plate cloning assay, we found that Nrp1 knockout significantly inhibited the proliferation of HCC cells. Nrp1 inhibitor suppressed lactate production and glucose consumption in HCC cells. Knockout of Nrp1 decreased the expression of glycolytic pathway-related proteins and HIF-1α protein. Furthermore, by joint use of CoCl2 and NRP1 knockout, we confirmed that reverting HIF-1α expression could reverse the effect of Nrp1 knockout on HCC cell metabolism in vitro. Mechanistically, Nrp1 showed a close correlation with the stability of HIF-1α protein in protein stability assay. Finally, we revealed that high expression of Nrp1 in HCC tissues was associated with poor overall survival and disease-free survival of the patients. Nrp1 accelerates glycolysis and promotes proliferation of HCC by regulating HIF-1α protein stability and through the VEGF/Nrp1/HIF-1α positive feedback loop.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Vascular Endothelial Growth Factor A/metabolism , Feedback , Neuropilin-1/genetics , Neuropilin-1/metabolism , Cell Proliferation , Glucose , Cobalt/pharmacology , Cobalt/metabolism , Lactates , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Cell Line, Tumor , Gene Expression Regulation, NeoplasticABSTRACT
BACKGROUND: Many studies have determined that dehydration is an independent predictor of outcome after ischemic stroke (IS); however, none have determined if the use of thrombolytic therapy modifies the negative impact of poor hydration. To inform the stroke registry established at our institution, we conducted a retrospective study to determine if dehydration remains a negative prognostic factor after IS patients treated with tissue plasminogen activator (tPA). METHODS: Between 2007 and 2012, we recruited 382 subjects; 346 had data available and were divided into 2 groups on the basis of their blood urea nitrogen/creatinine (BUN/Cr) ratio. Dehydrated subjects had a BUN/Cr ratio ≥ 15; hydrated subjects had a BUN/Cr < 15. The primary outcome was impairment at discharge as graded by the Barthel Index (BI) and the modified Rankin Scale (mRS). RESULTS: The dehydration group had a greater mean age; more women; lower mean levels of hemoglobin, triglycerides, and sodium; and higher mean potassium and glucose levels. A favorable outcome as assessed by the mRS (≤2) was significantly less frequent among dehydrated subjects, but a favorable outcome by the BI (≥60) was not. Logistic regression and multivariate models confirmed that dehydration is an independent predictor of poor outcome by both the mRS and the BI; however, it was not predictive when patients were stratified by Trial of Org 10,172 in Acute Stroke Treatment subtype. CONCLUSIONS: Our findings indicate that use of thrombolytic therapy does not eliminate the need to closely monitor hydration status in patients with IS.
