Fig. 3

tRF-5028c suppressed CRKL expression by directly targeting CRKL. A Prediction of tsRNA target genes and network construction based on miRanda and TargetScan. The red dots represent the eight upregulated tsRNAs, the green dots represent the two downregulated tsRNAs, and the pink dots represent their predicted target genes. B GO analysis of biological processes of tsRNA target genes. C KEGG analysis of signaling pathways of tsRNA target genes. D Venn diagram showing the intersection of tRF-5028c target genes with the Rap1 pathway genes. E Representative IHC images of villous expression and localization of CRKL in patients with RSA (n = 15) and healthy controls (n = 15). IgG is used as an isotype control, and the arrows indicate different types of trophoblasts (CTB, STB, and EVT). F Quantification of the percentage of CRKL-positive cells based on IHC staining. G The mRNA levels of CRKL in the villous tissues of patients with RSA (n = 15) and healthy controls (n = 15) were detected by qRT-PCR. H Pearson correlation analysis of tRF-5028c and CRKL mRNA levels in the villous tissues of patients with RSA (n = 15). I A 2D map of the potential binding sites between tRF-5028c and CRKL was obtained by integrating the algorithms of miRanda and TargetScan. The 5′ seed sequence of tRF-5028c (position 2–7, purple) is complementary to the 3′-UTR of CRKL mRNA (TGTGGG, blue). J The 3′-UTR of CRKL and the mutated sequence (shown in red) were cloned into the pGL3 vector to construct wild-type (WT) and mutant (MUT) reporter plasmids. Dual luciferase reporter gene assay was performed to analyze the interaction between tRF-5028c and CRKL. K, L HTR-8/SVneo cells were transfected with tRF-5028c inhibitor/mimic or inhibitor/mimic NC for 48 h, and the mRNA and protein levels of CRKL were detected by qRT-PCR and western blot, respectively. Data presented as mean ± SD. *p < 0.05, ***p < 0.001