Let-7i-5p Regulation of Cell Morphology and Migration Through Distinct Signaling Pathways in Normal and Pathogenic Urethral Fibroblasts

Abstract

microRNAs regulate subcellular functions through distinct molecular mechanisms. In this study, we used normal and pathogenic fibroblasts in pelvic fracture urethral distraction defects (PFUDD) patients. PFUDD is a common disease that could severely affect patients' life quality, yet little is known about the molecular mechanism associated with pathogenic fibrosis in PFUDD. Our data showed that let-7i-5p performs a multi-functional role in distinct signaling transduction pathways involved in cell morphology and cell migration in both normal and pathogenic fibroblasts. By analyzing the molecular mechanism associated with its functions, we found that let-7i-5p regulates through its direct target genes involved in collagen metabolism, cell proliferation and differentiation, TGF-beta signaling, DNA repair and ubiquitination, gene silencing and oxygen homeostasis. We conclude that let-7i-5p plays an essential role in regulating cell shape and tissue elasticity, cell migration, cell morphology and cytoskeleton, and could serve as a potential target for clinical treatment of urethral stricture patients.

Keywords: cell migration; cell morphology; fibroblast; let-7i-5p; microRNA.

FIGURE 1

Let-7i-5p is conservative among different species and hsa-let-7i-5p is expressed differentially in normal human tissues. By lenti-viral infection the let-7i-5p expression was manipulated either up or down in normal and scar tissues. (A) Let-7i-5p sequence comparison across different species. The conservative sequences are highlighted. aca, Anolis carolinensis; ami, Alligator mississippiensis; chi, Capra hircus; cli, Columba livia; cpi, Chrysemys picta; cpo, Cavia porcellus; dno, Dasypus novemcinctus; gmo, Gadus morhua; hsa, Homo sapiens; mdo, Monodelphis domestica; mmL, Macaca mulatta; mmu, Mus musculus; ocu, Oryctolagus cuniculus; oha, Ophiophagus hannah; pal, Pteropus alecto; pbv, Python bivittatus; rno, Rattus Norvegicus; ssa, Salmo Salar; ssc, Sus scrofa; tch, Tupaia chinensis; tgu, Taeniopygia guttata; xla, Xenopus laevis. (B) Hsa-let-7i-5p expression levels in normal human tissues. Data based on two individuals’ microRNA sequencing results (Ludwig et al., 2016) and average of normalized value by quantile normalization were used. (C) let-7i-5p level was up- and down-regulated in normal and pathogenic fibroblasts by Lenti-viral transfection. *p < 0.001. F, normal fibroblasts (HFF). S, scar tissues. NC, non-transfected control. SI, transfected by lenti-KD miRNA to knock down hsa-let-7i-5p expression. OE, transfected by lenti-OE miRNA to overexpress hsa-let-7i-5p.

FIGURE 2

Let-7i-5p level change results in cell morphology changes in normal fibroblasts, but not in pathogenic fibroblasts. Scale bar in bright field images, 100 μm. Scale bar in fluorescent images, 25 μm.

FIGURE 3

Overexpression of let-7i-5p could suppress migration, while inhibition could promote cell migration in both normal and pathogenic fibroblasts.

FIGURE 4

Let-7i-5p regulates signaling molecules in normal and pathogenic fibroblasts. Quantitative analysis of mRNA expression levels of let-7i-5p’s downstream targets were performed. Group 1, target genes positively regulated by hsa-let-7i-5p in normal fibroblast cells at mRNA level. Group 2, target genes negatively regulated by hsa-let-7i-5p in normal fibroblast cells at mRNA level. Group 3, target genes constitutively upregulated in normal and pathogenic fibroblasts at mRNA level with either suppressed or increased hsa-let-7i-5p expression. Error bar, standard error. mRNA level expression was evaluated by quantitative real-time PCR.

FIGURE 5

Let-7i-5p regulates MMP2 and TGF-beta1 proteins in normal and pathogenic fibroblasts (ELISA). Protein level expression was evaluated by ELISA.

FIGURE 6

Let-7i-5p regulation mechanism is summarized in distinct functional pathways. Direct targets were connected to let-7i-5p by golden lines.

FIGURE 7

Let-7i-5p regulates direct target genes involved in collagen metabolism, cell proliferation, TGFbeta signaling, DNA repair and ubiquitination, gene silencing and oxygen homeostasis. (A) Quantitative analysis of protein expression levels of target genes. Y axis represents the expression change normalized by FNC for each protein. (B) Representative western blot images.