Purpose: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)–overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen. Materials and Methods: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription–quantitative polymerase chain reaction. Results: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment. Conclusion The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

BACKGROUND: Fibrinogen is an essential cofactor for blood coagulation. The fibrinogen level has been identified as a risk factor for coronary artery disease(CAD) and stroke, but the relationship between plasma fibrinogen levels and number of atherosclerotic vesssels has been less investigated. The aim of this study is assess the possible association between plasma fibrinogen levels and the number of coronary stenosis in patients with CAD undergoing coronary catheterization. We also investigated the usefulness of plasma fibrinogen to predict CAD in a case-control study of the middle-aged men and women. METHODS: We measured plasma fibrinogen, total cholesterol, HDL-cholesterol and triglyceride in 121 patients with CAD and in 109 healthy controls. Multivariate logistic regression models were performed to assess risk factors for CAD. RESULTS: Plasma fibrinogen levels were significantly elevated in CAD group vs control group, 413.9+/-119.4 vs 296.3+/-74.1 mg/dL(P<0.001), respectively. The levels of plasma fibrinogen were not different according to the number of coronary stenosis. Multivariate logistic regression analysis of fibrinogen level and risk factors revealed 3 independent predictors of CAD: fibrinogen, body mass index and diabetes mellitus. Those with fibrinogen levels of 331-420 mg/dL had a 6.36-fold increased risk than fibrinogen levels of less than 270 mg/dL, while fibrinogen levels higher than 420 mg/dL had 3.53-fold increased risk. CONCLUSIONS: These results provide the evidence that plasma fibrinogen is associated with an increased risk of CAD. However, the plasma fibrinogen was not correlated with the severity of coronary stenosis.
Blood Coagulation ; Body Mass Index ; Case-Control Studies ; Catheterization ; Catheters ; Cholesterol ; Coronary Artery Disease* ; Coronary Stenosis ; Coronary Vessels* ; Diabetes Mellitus ; Female ; Fibrinogen* ; Humans ; Logistic Models ; Male ; Plasma* ; Risk Factors* ; Stroke ; Triglycerides