LncRNA-RMST Functions as a Transcriptional Co-regulator of SOX2 to Regulate miR-1251 in the Progression of Hirschsprung's Disease

Abstract

Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of enteric neural crest cells (ENCCs). LncRNA rhabdomyosarcoma 2-associated transcript (RMST) is essential for the growth and development of neuron. This study aimed to reveal the role of RMST in the pathogenesis of HSCR. The expression level of RMST, miR-1251, SOX2, and AHNAK was evaluated with qRT-PCR or western blot. CCK-8 and transwell assays were applied to detect cell proliferation and migration. CHIP and RIP assays were applied to determine the combination relationship between SOX2 and promoter region of miR-1251 or RMST and SOX2, respectively. Dual-luciferase reporter assay was performed to confirm miR-1251 targeted AHNAK. As results have shown, RMST was downregulated in the aganglionic colon of HSCR patients. The knockdown of RMST attenuated cell proliferation and migration significantly. MiR-1251, the intronic miRNA of RMST, was also low expressed in HSCR, but RMST did not alter the expression of miR-1251 directly. Furthermore, SOX2 was found to regulate the expression of miR-1251 via binding to the promoter region of miR-1251, and RMST strengthened this function by interacting with SOX2. Moreover, AHNAK was the target gene of miR-1251, which was co-regulated by RMST and SOX2. In conclusion, our study demonstrated that RMST functioned as a transcriptional co-regulator of SOX2 to regulate miR-1251 and resulted in the upregulation of AHNAK, leading to the occurrence of HSCR. The novel RMST/SOX2/miR-1251/AHNAK axis provided potential targets for the diagnosis and treatment of HSCR during embryonic stage.

Keywords: AHNAK; Hirschsprung's disease; SOX2; lncRNA-RMST; miR-1251.

Figure 1

RMST and miR-1251 were downregulated in HSCR patients. (A) RMST was downregulated obviously in aganglionic tracts of HSCR patients than control ones. (B) Transwell and CCK-8 assays showed that cell migration and proliferation was inhibited when RMST was knocked down in 293T and SH-SY5Y cells. (C) MiR-1251 was low expressed obviously in aganglionic colon tissues of HSCR patients compared with controls. (D) Cell proliferation and migration was attenuated obviously when miR-1251 was knocked down in 293T and SH-SY5Y cells. **p < 0.01, ***p < 0.001.

Figure 2

miR-1251 was transcriptionally regulated by SOX2. (A) CHIP assay showed that SOX2 could bind to the promoter region of miR-1251. (B) After downregulating SOX2, the expression of miR-1251 was reduced. (C,D) SOX2 was downregulated in HSCR patients at both mRNA and protein level. (E–G) When 293T and SH-SY5Y cells were transfected with si-SOX2, cell migration, and proliferation was inhibited, but the upregulation of miR-1251 could partially reverse it. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

RMST functioned as a co-regulator of SOX2. (A) RIP assay confirmed RMST could bind to SOX2 protein. (B) The expression of miR-1251 was downregulated in “SOX2 siRNA” group and was reduced more in “RMST siRNA+SOX2 siRNA” group, but was not changed significantly in “RMST siRNA” group compared with the control group. (C,D) CCK-8 and transwell assays revealed that when the expression of RMST and SOX2 were both knocked down, the cell proliferation and migration was more weakened than just downregulated RMST or SOX2 alone. However, the upregulation of miR-1251 could partly reverse the inhibitory effects of “RMST siRNA+SOX2 siRNA” on cell proliferation and migration. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

AHNAK was the target gene of miR-1251. (A) The schematic diagram of binding sites between miR-1251 and AHNAK. (B) The luciferase activity was abated obviously when transfected with miR-1251 mimics and pLG3-AHNAK-wild compared with control; however, the luciferase activity was not changed significantly when transfected with miR-1251 mimics and pLG3-AHNAK-mut in 293T and SY5Y cells. (C) When miR-1251 was knocked down, the mRNA level of AHNAK was raised. (D,E) AHNAK was upregulated in stenotic tracts of HSCR patients at mRNA and protein level compared with control tracts. (F–H) The downregulation of miR-1251 inhibited cell migration and proliferation, but the knockdown of AHNAK could partially reverse it in 293T and SH-SY5Y cells. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

RMST functioned as a SOX2 transcriptional co-regulator to inhibit miR-1251 and raise AHNAK expression. (A,B) When cells were transfected with RMST siRNA, the mRNA, and protein expression of AHNAK was not changed obviously; however, the transfection of SOX2 siRNA raised the expression of AHNAK and the downregulation of both RMST and SOX2 increased this more. (C,D) The cell proliferation and migration was inhibited in “RMST siRNA” or “SOX2 siRNA” groups compared with control and was inhibited more obviously in “RMST siRNA+SOX2 siRNA” group. (E) However, the simultaneous downregulation of AHNAK could partially alleviate the inhibitory effects caused by the knockdown of both RMST and SOX2. ns, p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.