Fig. 7: GELN-enriched with aly-miR159a-3p modulates metabolism of DHA in bacteria by targeting PLC.

A Relative level of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in LI and SI of GF and hFB mice, respectively, using HPLC (n = 3). (P = 0.0001, P = 0.0022; P = 0.0002, P = 0.0079, two-way ANOVA test, n = 3). B HPLC analysis of DHA and EPA in the gut feces from GF and hFB mice as well as medium of Clostridium perfringens (C. perf) (C. perf, ATCC 13124) treated with GNV-RNAs (0.5 mg/kg, body weight; 10 mg/1 × 108 bacteria) and ELN-RNAs mixed from garlic, aloe, and lemon. C Top five abundance miRNAs in GELN from NGS miRNA sequencing. D Schematic diagram of the putative binding sites of aly-miR159a-3p in the phospholipase C (PLC) of C. perf. Red arrows indicate primers for ChIP. The pink bar indicates the lipoxygenase domain. The aly-miR159a-3p seed matches in the PLC gene mutated at the positions indicated. E Schematic representation of DHA metabolism derived from EPA and α-linolenic acid (ALA) and converted to 4-oxo-DHA by PLC. The hypothetical model of GELN-derived aly-miR159a-3p regulates the metabolism of DHA and EPA by targeting PLC. F Analysis of interaction between miR159a-3p and PLC gene using the ChIP assay. Biotin-labeled aly-miR159a-3p incubated with DNA from C. perf (left panel) and feces from the colonized hFB mouse (right panel). PLC gene binding to aly-miR159a-3p pulled down with streptavidin beads and amplified with specific PLC primers. G qPCR analysis of PLC expression in C. perf treated with scrambled miRNA, aly-miR159a-3p, and miR159a mutant. (P = 0.0012, two-way ANOVA test, n = 3). H Western blot analysis of PLC expression. C. perf enterotoxin (CPE) served as a loading control. The size (kDa) of protein molecular weight (MW) indicated. I HPLC analysis of EPA (left), DHA (middle), and 4-oxo-DHA (right) in C. perf treated with aly-miR159a as well as control miRNAs. (P = 0.4478, P = 0.0074, P = 0.0062, two-way ANOVA test, n = 3). J Schematic diagram of PLC knockout (KO) in C. perf and replaced with ampicillin-resistant (Amp⁺) sequence using fusion PCR (left panel). Western blot analysis for wildtype (WT) and PLC KO C. perf (right panel). CPE was the loading control. The size (kDa) of protein MW indicated. K HPLC analysis of DHA in WT and PLC KO C. perf. (P = 0.0073, P = 0.0034, two-way t test, n = 3). Data are representative of three independent experiments as the mean ± SD (error bars). Source data are provided as a Source Data file.