ABSTRACT
Objective:To establish a performance validation method for mNGS applied in BALF samples.Method:Hela cells were used as a representative of host cells, and simulated BALF samples were prepared by adding different concentrations of Hela cells, seven species of isolated pathogens (including Streptococcus pneumonia, Hemophilus influenza, Klebsiella pneumonia, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Adenovirus), and interfering substances to sterile normal saline. Clinical BALF samples were collected simultaneously, and the results of mNGS were evaluated using traditional detection methods as a reference. The limit of detection (LOD), precision, anti-interference ability, stability, and accuracy of mNGS were determined. Results:In the simulated samples, the LOD of Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Adenovirus were 150, 262, 102, 67, 96, 83 CFU/ml, and 439 copies/ml, respectively. The repeatability of the detection results for all pathogens of simulated positive BALF samples was 100%. The anti-interference test showed that the higher the concentration of human DNA, the fewer pathogen sequences detected by mNGS. Escherichia coli and Shigella sonnei were used to evaluate the ability of mNGS to distinguish closely related species. The results showed that the system could stably distinguish Escherichia coli and Shigella sonnei when the concentration of Shigella sonnei was 4, 000 CFU/ml. The stability test results showed that there was no significant change in the number of pathogen sequences detected whether after 1 to 3 freeze-thaw cycles or storage at 4 ℃, -20 ℃, or -80 ℃ for 36 h. Compared with traditional detection methods, the accuracy of 17 clinical samples was 82.4%(14/17). Continuous evaluation of clinical BALF samples simultaneously tested by mNGS and traditional methods at Tongji Hospital from October 25, 2021, to September 14, 2022, showed that the accuracy of mNGS compared to bacterial culture, fungal culture, mycobacterial culture, Mycobacterium tuberculosis culture, and conventional PCR techniques was 67.5%(472/699), 81.5%(570/699), 92.3%(335/363), 96.4%(350/363), and 86.8%(132/152), respectively. Compared with conventional PCR techniques, the accuracy of mNGS for detecting Pneumocystis jirovecii, Adenovirus, and Mycoplasma pneumoniae was 89.4%(84/94), 93.3%(56/60), and 87.1%(61/70), respectively. Conclusion:By preparing simulated BALF samples and using traditional detection methods as a reference, the performance characteristics of mNGS in detecting BALF samples can be preliminarily evaluated.
ABSTRACT
Objective:To investigate the expression of microRNA (miR) -26a in human skin fibroblasts during photoaging induced by ultraviolet A (UVA) , and to evaluate the effect of up-or down-regulation of miR-26a expression on the methylation level of the whole genome, the target gene enhancer of zeste homolog 2 (EZH2) and cell aging.Methods:Some human skin fibroblasts were irradiated with 10 J/cm 2 UVA once a day for 7 consecutive days, RNA was extracted on days 0, 3 and 7, and real-time quantitative reverse PCR (RT-PCR) was performed to determine the expression of miR-26a; miR-26a mimics and inhibitors were transfected into fibroblasts to up-or down-regulate the expression of miR-26a respectively, and fluorescence microscopy and RT-PCR were performed to determine the expression of miR-26a and evaluate the transfection efficiency. Some human skin fibroblasts were divided into 6 groups: blank control group receiving no treatment, UVA group treated with UVA irradiation according to the above method, miR-26a mimic group transfected with miR-26a-mimics, UVA+miR-26a mimic group transfected with miR-26a-mimics followed by UVA irradiation, miR-26a inhibitor group transfected with miR-26a inhibitors, UVA+miR-26a inhibitor group transfected with miR-26a inhibitors followed by UVA irradiation. On day 7, cells in each group were collected after the end of UVA irradiation. Then, flow cytometry was performed to detect cell cycle, DNA methylation quantitative detection kit was used to detect the methylation level of whole genome, RT-PCR was conducted to determine the mRNA expression of EZH2 (a histone-lysine N-methyltransferase enzyme) , DNA methyltransferase 1 (DNMT1) and miR-26a, and Western blot analysis was performed to determine the protein expression of EZH2 and DNMT1. Statistical analysis was carried out by using one-way analysis of variance and least significant difference- t test. Results:Compared with the unirradiated control group, the expression of miR-26a gradually increased in the UVA irradiation group over time during the culture, and there was a significant difference in the expression of miR-26a between the two groups after 7 days of UVA irradiation ( t=5.295, P < 0.05) . Strong fluorescence signals were observed in the miR-26a mimic-or miR-26a inhibitor-transfected fibroblasts, suggesting a high transfection efficiency. Flow cytometry showed that the proportion of cells at G1 phase significantly differed among the blank control group, UVA group, miR-26a mimic group, UVA+miR-26a mimic group, miR-26a inhibitor group, and UVA+miR-26a inhibitor group (52.82% ± 2.56%, 78.56% ± 4.34%, 53.63% ± 3.13%, 89.52% ± 4.17%, 54.39% ± 3.86%, 65.34% ± 4.78%, respectively; F=46.728, P < 0.01) , and significantly higher in the UVA group than in the blank control group ( t=8.848, P < 0.01) , higher in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group ( t=11.922, 3.154, P < 0.01, < 0.05, respectively) , and higher in the UVA+miR-26a inhibitor group than in the miR-26a-inhibitor group ( t=3.087, P < 0.05) , but significantly lower in the UVA+miR-26a inhibitor group than in the UVA group ( t=3.547, P < 0.05) . Detection of the genome-wide methylation level showed that the methylation level ( A450 value) significantly differed among the above groups (0.676 ± 0.024, 0.323 ± 0.043, 0.506 ± 0.035, 0.169 ± 0.024, 0.602 ± 0.036, 0.422 ± 0.029, respectively, F=97.402, P < 0.01) , and significantly lower in the UVA group than in the blank control group ( P < 0.01) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.01) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.01) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . RT-PCR and Western blot analysis showed significant differences in the mRNA and protein expression of EZH2 and DNMT1 respectively among the 6 groups (both P < 0.05) , which were significantly lower in the UVA group than in the blank control group ( P < 0.05) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.05) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.05) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . Conclusion:In the UVA irradiation-induced photoaging of skin fibroblasts, miR-26a expression was up-regulated, cellular proliferative activity and genome-wide methylation level decreased; up-regulation of miR-26a expression could down-regulate the expression of its target gene EZH2 and methylation-related gene DNM1, and promote cell photoaging, while down-regulation of miR-26a expression could up-regulate the expression of EZH2 and DNMT1, and inhibit cell photoaging.