MiR-195-5p inhibits proliferation and invasion of nerve cells in Hirschsprung disease by targeting GFRA4

Studies have reported that miR-195-5p plays a role in the Hirschsprung disease (HSCR). Our previous work found GDNF family receptor alpha 4 (GFRA4) is also associated with HSCR. In this study, we focused on whether miR-195-5p induces the absence of enteric neurons and enteric neural crest in HSCR by regulating GFRA4. The expression levels of GFRA4 and miR-195-5p in colon tissues were evaluated by real-time PCR (RT-PCR) assay. We overexpressed GFRA4 or miR-195-5p in SH-SY5Y cells, the cell proliferation, cell cycle, apoptosis and invasion were subsequently investigated by CCK-8 assay, EdU staining, Flow cytometry analysis and Transwell assay, respectively. We also established the xenograft model to detect the effect of miR-195-5p on tumor growth and GFRA4 and p-RET expressions. GFRA4 expression was significantly downregulated in the HSCR colon tissues when compared with that in the control tissues. Overexpression of GFRA4 significantly promoted proliferation, invasion and cell cycle arrest, and inhibited apoptosis of SH-SY5Y cells. We also proved that GFRA4 is a direct target of miR-195-5p, and miR-195-5p inhibited proliferation, invasion, cell cycle arrest and differentiation, and accelerated apoptosis in SH-SY5Y cells which can be reversed by GFRA4 overexpression. Furthermore, we demonstrated that miR-195-5p suppressed tumor growth, and observably decreased GFRA4 and p-RET expressions. Our findings suggest that miR-195-5p plays an important role in the pathogenesis of HSCR. MiR-195-5p inhibited proliferation, invasion and cell cycle arrest, and accelerated apoptosis of nerve cells by targeting GFRA4.