Let-7c increases BACE2 expression by RNAa and decreases Aβ production

Abstract

MicroRNAs (miRNAs) are highly conserved, non-coding transcripts that regulate gene expression in various ways. Evidence suggests that miRNAs may be a contributory factor in neurodegeneration, including Alzheimer's disease (AD), Parkinson's disease (PD), and triplet repeat disorders. In order to further understand the potential roles of miRNAs in the pathogenesis of AD, we analyzed Down syndrome (DS), a special model of AD, by using a TaqMan microRNA array and found that miRNA let-7c was up-regulated in both DS and AD. ELISA assay showed that let-7c reduced the expression level of Aβ significantly. Real-time quantitative-polymerase chain reaction (RT-qPCR) was conducted to reveal that the expression level of let-7c increased dramatically in DS cells, patients with DS and mice with AD compared with normal ones respectively. Additionally, western blotting illustrated that let-7c suppressed the expression of Aβ by inducing BACE2 to cut C99 and increase the content of C83/80. BACE2 expression was inhibited by let-7c and luciferase reporter gene assay revealed that let-7c increased the activity of wild-type BACE2 promoter but not 3'UTR. Furthermore, promoter analysis of BACE2 confirmed that let-7c could bind to BACE2 in the sequence between -1368 and -1347. In addition, immunoblotting assay demonstrated that let-7c induced BACE2 expression by RNAa. To the best of our knowledge, our study revealed for the first time that let-7c up-regulated BACE2 expression and decreased Aβ production.

Keywords: Alzheimer’s disease; BACE2; Let-7c; RNA activation; β-amyloid.

Figure 1

Let-7c increased in DS cell line and reduced generation of Aβ. A. Low-density Taqman array performed to identify panels of miRNA expression in DS cell line MB1478 and normal cell line UMB115; B. 20E2 cells transfected with pmiR-let-7c, pmiR-99a, pmiR-155 or control, and Aβ quantity detected by ELISA Assay. *P<0.05, let-7c compared to the control group; C and D. pZ-APPsw and pAPP-C99 transfected into HEK293 cells with pmiR-let-7c or not, respectively, and Aβ quantity detected by ELISA Assay. *P<0.05, compared to the control group; E. HEK293 cells transfected with pmiR-let-7c or not, respectively, and cell proliferation detected by MTT assay. Values represent means ± SD, n=3.

Figure 2

Let-7c expression panels in DS cell lines, DS patients and AD mice. A. pre-let-7c expression detected by RT-PCR in DS cell line MB1478 and normal cell line UMB115. *P<0.05, compared to the normal group; B. Mature let-7c expression detected by RT-qPCR in cell lines MB1478 and UMB115. *P<0.05, compared to the normal group; C and D. let-7c expression detected by RT-qPCR in DS patients (Patients =8, Normal =5) and AD mice (Models =12, Normal =8), respectively. *P<0.05, compared to the normal group. Values represent means ± SD, n=3.

Figure 3

let-7c reduces Aβ production by inducing BACE2 to cut C99. A. 2EB2 cells transfected with pmiR-let-7c or not, and C99 and C83/C80 protein detected by C20 antibody. *P<0.05, let-7c compared to the control group; B. pAPP-C99Flag transfected into HEK293 cells with pmiR-let-7c or not, and C99 and C83/C80 protein detected by C20 antibody. *P<0.05, let-7c compared to the control group; C. pAPP-C99, pAPP-C99 and pshBACE2, pAPP-C99 and pshNCT transfected into HEK293 cells with pmiR-let-7c or not, and C99 protein detected by C20 antibody. *P<0.05, let-7c compared to the control group; D. pAPP-C83, pAPP-C83 and pshBACE2, pAPP-C83 and pshNCT transfected into HEK293 cells with pmiR-let-7c or not, and C83 protein detected by C20 antibody. *P<0.05, let-7c compared to the control group; E. HEK293 cells transfected with pKv2.1-Flag and pmiR-let-7c or not, and KCNB1 protein detected by M2 antibody. *P<0.05, compared to the control group. Values represent means ± SD, n=3.

Figure 4

Let-7c targeted BACE2 promoter. A. HEK293 cells transfected with pmiR-let-7c or not, and BACE2 expression detected by RT-qPCR. *P<0.05, compared to the control group; B-F. pB2Luc-A, pBACE2-3UTRluc, pB1P-A, pBACE1-3UTRluc and prhβAPPluc transfected into HEK293 cells with pmiR-let-7c or not, respectively, and dual luciferase assay performed 48 hours after transfection. *P<0.05, compared to the control group. Values represent means ± SD, n=3.

Figure 5

Let-7c induces BACE2 expression through RNAa. A. Schematic diagram of the structure of BACE2 promoter and three deleted constructs; B. pB2Luc-A, pB2Luc-B, pB2Luc-C and pB2Luc-D transfected into HEK293 cells with pmiR-let-7c or not, respectively, and dual luciferase assay performed 48 hours after transfection. *P<0.05, compared to the pGL3-Basic group; C. Sequence alignment of BACE2 and let-7c, with sequences of BACE2 and let-7c aligned using ClustalW2 software; D. HEK293 cells transfected with pshAGO1 or pshAGO2, and AGO1 or AGO2 expression detected by RT-PCR. *P<0.05, compared to the control group; E. HEK293 cells transfected with pshAGO1 or pshAGO2, and BACE2 expression detected by RT-PCR. *P<0.05, compared to the si-control group; F. pAPP-C99Flag and pmiR-let-7c transfected into HEK293 cells with pshAGO1 or pshAGO2, and C99 protein detected by M2 antibody. *P<0.05, compared to the si-control group. Values represent means ± SD, n=3.

Figure 6

Schematic diagram of miRNA let-7c inducing BACE2 expression and inhibiting Aβ production.