[Inhibition of Ferulic Acid on U937 Cell Growth and Its Related Mechanism].

OBJECTIVE: To investigate the effects of ferulic acid (FA) on proliferation, apoptosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway of human acute myeloid leukemia (AML) U937 cells. METHODS: Different concentrations of FA (0、10、25、50、100、200 µmol/L) was used to treat human AML cell lines Kasumi-1, HL-60, and U937 cells respectively for 24 h. The cell survival rate was detected by cell counting kit-8 method, and the 50% inhibitory concentration (IC50) of each cell line was calculated. The U937 cells were divided into control group, FA group (50 µmol/L FA), FA+pcDNA group (50 µmol/L FA+transfected with empty plasmid), FA+pcDNA-TLR4 group (50 µmol/L FA+transfected with TLR4 overexpression plasmid). Flow cytometry was used to detect U937 cell cycle and cell apoptosis; plate clone formation test was used to detect U937 cell clone formation ability; fluorescence quantitative PCR was used to detect the TLR4, NF-κB p65 mRNA levels in U937 cells; Western blot was used to detect the expression levels of CyclinD1, CyclinE, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2), Caspase-3, TLR4, and NF-κB p65 protein in U937 cells. RESULTS: With the increase of FA concentration, the survival rates of Kasumi-1, HL-60 and U937 cells gradually decreased(r=-0.919, r=-0.909, r=-0.900), the IC50 of U937 cells was 50.25±2.23 µmol/L. Compared with the control group, after drug treatment of U937 cells, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA group were significantly increased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and protein were significantly decreased (P<0.05); compared with FA group and FA+pcDNA group, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA+pcDNA-TLR4 group were significantly decreased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and proteins were significantly increased (P<0.05). CONCLUSION: FA inhibits U937 cell proliferation and promotes cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway.

C-reactive protein correlates with computed tomographic findings and predicts severe COVID-19 early.

COVID-19 has developed into a worldwide pandemic; early identification of severe illness is critical for controlling it and improving the prognosis of patients with limited medical resources. The present study aimed to analyze the characteristics of severe COVID-19 and identify biomarkers for differential diagnosis and prognosis prediction. In total, 27 consecutive patients with COVID-19 and 75 patients with flu were retrospectively enrolled. Clinical parameters were collected from electronic medical records. The disease course was divided into four stages: initial, progression, peak, and recovery stages, according to computed tomography (CT) progress. to mild COVID-19, the lymphocytes in the severe COVID-19 progressively decreased at the progression and the peak stages, but rebound in the recovery stage. The levels of C-reactive protein (CRP) in the severe group at the initial and progression stages were higher than those in the mild group. Correlation analysis showed that CRP (R = .62; P < .01), erythrocyte sedimentation rate (R = .55; P < .01) and granulocyte/lymphocyte ratio (R = .49; P < .01) were positively associated with the CT severity scores. In contrast, the number of lymphocytes (R = -.37; P < .01) was negatively correlated with the CT severity scores. The receiver-operating characteristic analysis demonstrated that area under the curve of CRP on the first visit for predicting severe COVID-19 was 0.87 (95% CI 0.10-1.00) at 20.42 mg/L cut-off, with sensitivity and specificity 83% and 91%, respectively. CRP in severe COVID-19 patients increased significantly at the initial stage, before CT findings. Importantly, CRP, which was associated with disease development, predicted early severe COVID-19.

Tumor-targeted efficiency of shRNA vector harboring chimera hTERT/U6 promoter.

Telomerase is closely related to tumor, and hTERT is the rate-limiting factor for telomerase activity. The transcription and expression of hTERT is determined by hTERT promoter, which has the ability of anchoring telomerase positive cells. RNA interference (RNAi) has been potentially used in the functional genomics and gene therapy recently. However, the limitations of RNAi uncertain interference and safety hamper its wide applications. To overcome these limitations, we constructed shRNA vectors harboring either U6 promoter or chimera hTERT/U6 promoter aiming at EGFP and hTERT genes (shRNA-EGFP-U6, shRNA-EGFP-hTERT/U6, shRNA-hTERT-U6 and shRNA-hTERT-hTERT/U6), to suppress the expression of GFP and hTERT in telomerase negative human normal fibroblast HELF cells and telomerase positive human hepatocarcinoma SMMC-7721 and HepG2 cells, respectively. HELF-EGFP and SMMC-7721-EGFP cells stably expressing EGFP or hTERT were constructed. GFP expression was inhibited in both HELF-EGFP and SMMC-7721-EGFP cells expressing shRNA-EGFP-U6. Further results showed that GFP expression was suppressed only in telomerase positive SMMC-7721 cells and HepG2 cells, but not in telomerase negative HELF cells expressing shRNA-EGFP-hTERT/U6. Further results found that hTERT expression was effectively inhibited from liver cancer cells expressing shRNA-hTERT-U6 or shRNA-hTERT-hTERT/U6 both in vitro and in vivo. Our study illustrates the tumor-targeted efficiency of shRNA vectors harboring chimera hTERT/U6 promoter in telomerase positive cells, which will benefit tumor therapy.