[Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis].

OBJECTIVE: To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis. METHODS: (1) Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. (2) Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting. RESULTS: (1) Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (µg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×109/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients (n = 20) was significantly higher than that in the non-thrombocytopenia patients (n = 37; µg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients (r = -0.340, P = 0.010). (2) Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×109/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/ß-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. CONCLUSIONS: PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.

LncRNA HOTAIRM1 knockdown inhibits cell glycolysis metabolism and tumor progression by miR-498/ABCE1 axis in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major contributor of cancer-related mortality. Long non-coding RNAs (lncRNAs) are indicated to participate in the pathogenesis of NSCLC. OBJECTIVE: In this research, the effects of lncRNA HOXA transcript antisense RNA, myeloid-specific 1 (HOTAIRM1) on NSCLC progression and underlying mechanism were revealed. METHODS: The expression levels of HOTAIRM1 and microRNA-498 (miR-498) were detected by quantitative real time polymerase chain reaction (qRT-PCR) in NSCLC tissues, cells or exosomes. The protein expression of CD63, CD81, hexokinase 2 (HK2) and ATP binding cassette subfamily E member 1 (ABCE1) was determined by western blot. Cell viability, apoptosis, migration and invasion were investigated by cell counting kit-8 (CCK-8), flow cytometry, transwell migration and invasion assays, respectively. Cell glycolysis metabolism was revealed by glucose uptake and lactate production assays and western blot analysis. The binding relationship between miR-498 and HOTAIRM1 or ABCE1 was predicted by DIANA-LncBase v2 and starBase online database, and identified by dual-luciferase reporter assay. The effects of HOTAIRM1 on NSCLC growth in vivo were revealed by in vivo tumor formation assay. RESULTS: HOTAIRM1 expression was dramatically upregulated, whereas miR-498 expression was significantly downregulated in NSCLC tissues cells or exosomes as compared to control groups. Mechanistically, HOTAIRM1 knockdown repressed cell viability, migration, invasion and glycolysis metabolism, whereas induced cell apoptosis in NSCLC; however, miR-498 inhibitor hindered these effects. Functionally, HOTAIRM1 functioned as a sponge of miR-498 and miR-498 targeted ABCE1. In addition, HOTAIRM1 silencing inhibited NSCLC growth in vivo by downregulating ABCE1 and upregulating miR-498 expression. CONCLUSIONS: HOTAIRM1 knockdown repressed cell glycolysis metabolism and tumor development by reducing ABCE1 expression through sponging miR-498 in NSCLC, which provided a theoretical basis for further studying NSCLC progression.