Sepsis patients showed altered expression of circFADS2 and miR-133a
RNA samples isolated from both sepsis patients (n = 62) and healthy controls (n = 62) were subjected to RT-qPCR to analyze differential gene expression in sepsis. Our data revealed that, compared to the 62 healthy controls, circFADS2 was under-expressed in sepsis (Fig. 1A, p < 0.001) and miR-133a was overexpressed (Fig. 1B, p < 0.001) in sepsis. Therefore, the downregulation of circFADS2 and the overexpression of miR-133a may participate in sepsis.
RNA samples isolated from both sepsis patients (n = 62) and healthy controls (n = 62) were subjected to RT-qPCR to analyze the differential expression of circFADS2 (A) and miR-133a (B) in sepsis. Ct values were normalized to corresponding internal controls following 2−ΔΔCT method. The sample with the biggest ΔCT value was set to value “1”. Other samples were normalized to this sample to calculate relative gene expression. ***,p < 0.001.
Plasma samples from sepsis patients showed an inverse correlation between circFADS2 and miR-133a
The differential expression pattern of circFADS2 and miR-133a in sepsis may indicate the potential crosstalk between them. To explore the interaction between them, the first step is to analyze the correlations. Pearson’s correlation coefficient analysis revealed that circFADS2 and miR-133a were inversely and significantly correlated across sepsis samples (Fig. 2A). However, correlation analysis revealed no close correlation between circFADS2 and miR-133a across control samples (Fig. 2B). The close correlation between them indicates the potential interaction between them.
The differential expression pattern of circFADS2 and miR-133a in sepsis may indicate the potential crosstalk between them. To explore the interaction between them, Pearson’s correlation coefficient analysis was performed to analyze the correlations between circFADS2 and miR-133a across sepsis samples (A) and control samples (B).
Overexpression of circFADS2 decreased the expression of miR-133a in HBEpCs
Expression vector of circFADS2 and the mimic of miR-133a were transfected into HBEpCs (pre-treated with 12 µg/ml LPS for 48 h) to further explore the crosstalk between circFADS2 and miR-133a. Overexpression of circFADS2 and miR-133a was analyzed by RT-qPCR every 24 h until 96 h. It was observed that circFADS2 and miR-133a were overexpressed as each time point (Fig. 3A, p < 0.05). It was observed that circFADS2 overexpression decreased the expression of miR-133a (Fig. 3B, p < 0.05). However, overexpression of miR-133a failed to significantly alter the expression of circFADS2 at each time point (Fig. 3C). Therefore, circFADS2 may downregulate miR-133a in HBEpCs.
Expression vector of circFADS2 and the mimic of miR-133a were transfected into HBEpCs (pre-treated with 12 µg/ml LPS for 48 h) to further explore the crosstalk between circFADS2 and miR-133a (A). The effects of circFADS2 overexpression on the expression of miR-133a (B) and the effects of miR-133a overexpression on the expression of circFADS2 (C) were also analyzed by RT-qPCR. *,p < 0.05.
LPS treatment altered the expression of circFADS2 and miR-133a in HBEpCs
LPS treatment was performed by incubating HBEpCs in medium containing 0, 2, 4, 8 and 12 µg/ml LPS under the aforementioned conditions for 48 h, followed by detecting the expression of circFADS2 and miR-133a through RT-qPCR. It was observed that LPS treatment decreased the expression of circFADS2 (Fig. 4A, p < 0.05) and increased the expression of miR-133a (Fig. 4B, p < 0.05). Therefore, the altered expression of circFADS2 and miR-133a in sepsis is likely induced by LPS.
LPS treatment was performed by incubating HBEpCs in medium containing 0, 2, 4, 8 and 12 µg/ml LPS under the aforementioned conditions for 48 h, followed by detecting the expression of circFADS2 (A) and miR-133a (B) through RT-qPCR. *,p < 0.05.
Overexpression of circFADS2 suppressed the apoptosis of HBEpCs induced by LPS through miR-133a
The effects of the overexpression of circFADS2 and miR-133a on the apoptosis of HBEpCs induced by LPS were analyzed by cell apoptosis assay. Our data showed that circFADS2 overexpression decreased cell apoptosis and miR-133a overexpression increased cell apoptosis. In addition, circFADS2 overexpression reduced the enhancing effects of miR-133a overexpression on cell apoptosis induced by LPS (Fig. 5, p < 0.05).
The effects of the overexpression of circFADS2 and miR-133a on the apoptosis of HBEpCs induced by LPS were analyzed by cell apoptosis assay.*,p < 0.05.