Suppressive action of miRNAs to ARP2/3 complex reduces cell migration and proliferation via RAC isoforms in Hirschsprung disease

Abstract

Hirschsprung disease (HSCR) is a congenital disorder caused by the defective function of the embryonic enteric neural crest. The impaired migration of embryonic enteric neural crest plays an important role in the pathogenesis of this disease. Recent studies showed that the ARP2/3 complex and RAC isoforms had effects on actin cytoskeleton remodelling, which contributes to migration. Moreover, some regulatory relationships were identified between ARP2/3 complex and RAC isoforms. Although microRNAs (miRNAs) have been known to modulate target gene expression on the post-transcriptional level, little is known about the regulation among miRNAs, ARP2/3 complex and RAC isoforms. Here, we report that down-regulation of ARP2 and ARP3, two main subunits of ARP2/3 complex, suppressed migration and proliferation in 293T and SH-SY5Y cell lines via the inhibition of RAC1 and RAC2. Meanwhile, as the target genes, ARP2 and ARP3 are reduced by increased miR-24-1* and let-7a*, respectively, in 70 HSCR samples as compared with 74 normal controls. Co-immunoprecipitation showed that aberrant reduction in ARP2 and ARP3 could weaken the function of ARP2/3 complex. Our study demonstrates that the miR-24-1*/let-7a*-ARP2/3 complex-RAC isoforms pathway may represent a novel pathogenic mechanism for HSCR.

Keywords: ARP2/3 complex; Hirschsprung disease; RAC isoforms; gene regulation; microRNA.

Figure 1

The expression level of ARP2, ARP3 and RAC isforms in HSCR/control tissues. (A) The mRNA expression level of ARP2 and ARP3 in control/HSCR tissues (**P = 0.0043, *P = 0.0256, n = 74 controls/70 HSCR, Mann–Whitney test). (B) The protein expression level of ARP2, ARP3, RAC1, RAC2 and RAC3 in HSCR/control tissues (*P < 0.05, **P < 0.01, n = 3, Unpaired t‐test). (C) The mRNA expression level of RAC1, RAC2 and RAC3 in control/HSCR tissues (**P = 0.0051, ***P = 0.0005, n = 74 controls/70 HSCR, Mann–Whitney test). All tests were performed for three times and presented as mean ± S.E.M.

Figure 2

The CO‐IP results and RAC1/2 were inhibited by upstream regulators. (A) The co‐immunoprecipitation results of ARP2 and ARP3 in 293T cell line. The cells were pre‐treated with the siRNAs of ARP2 or ARP3 and endogenous protein–protein interaction between ARP2 and ARP3 was demonstrated by immunoprecipitation (IP) with ARP2 or ARP3 antibodies. IgG was used as negative control for IP. (B) The mRNA and protein expression levels of RAC1 and RAC2 after the transfection of siRNA‐ARP2, siRNA‐ARP3, miR‐24‐1* mimics and let‐7a* mimics in 293T and SH‐SY5Y cell lines (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3, Unpaired t‐test). All tests were performed for three times and presented as mean ± S.E.M.

Figure 3

Cytobiology change after transfecting cell lines with RNA oligos. (A) The representative images of metastasis cells at the bottom of the membrane stained with crystal violet were visualized as shown (left). The quantifications of cell migration were presented as percentage migrated cell numbers and the integrated intensity of migrated cells (right) (*P < 0.05, **P < 0.01, ***P < 0.001, n = 5, Unpaired t‐test). (B) The result of CCK‐8 assay of 450 nm absorption (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6, Unpaired t‐test). All tests were performed for three times and presented as mean ± S.E.M.

Figure 4

Up‐regulated miR‐24‐1* and let‐7a* respectively inhibited ARP2 and ARP3 in double cell lines. (A) The expression level of miR‐24‐1* and let‐7a* in HSCR/control tissues (***P ≤ 0.0003, n = 74 controls/70 HSCR, Mann–Whitney test). (B) The binding sequence of miR‐24‐1*&ARP2 wild‐type, let‐7a*& ARP3 wild‐type and the sequence of mutant type of ARP2 and ARP3 in 3′‐UTR. (C) The results of luciferase reporter assay (**P < 0.01, ***P < 0.001, n = 8, Mann–Whitney test). (D) The mRNA and protein expression level of ARP2 and ARP3 after ilka transfection of miR‐24‐1* and let‐7a* mimics in 293T and SH‐SY5Y cell lines (**P < 0.01, ***P < 0.001, n = 6, Unpaired t‐test). All tests were performed for three times and presented as mean ± S.E.M.

Figure 5

Schematic representation of the miR‐24‐1*/let‐7a*‐ARP2/3 complex‐RAC isoforms pathway in HSCR.